适于人表皮组织蛋白质组分析的双向电泳技术  

Two-dimensional Electrophoresis for Proteomics Analysis of Human Epidermis

作　　者：聂燕芳[1] 曾耀英[1] 吴晓萍[1] 刘钧澄[2] 黄秀艳[1] 

机构地区：[1]暨南大学组织移植与免疫中心,广东广州510632 [2]中山大学附属第一医院小儿外科,广东广州510080

出　　处：《中山大学学报（医学科学版）》2008年第3期337-341,共5页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家重点基础研究规划项目973计划项目(2004CB720100);国家重点基础研究发展计划973项目(2006CB504201)

摘　　要：【目的】建立适于人表皮组织的双向电泳技术,并应用质谱技术对人表皮组织蛋白质组进行初步分析。【方法】利用组织匀浆法制备人表皮组织总蛋白、固相pH梯度胶条进行双向电泳、PDQuest7.4软件比较和分析电泳图谱,利用质谱技术鉴定部分蛋白斑点。【结果】成功建立了适用于人表皮组织蛋白质组分析的双向电泳技术,获得和比较了人表皮组织双向电泳四种染色图谱。人表皮组织的蛋白质在pH5～8范围内分布均匀,分子量集中在15～100ku之间。银染图谱获得的蛋白斑点数为586±14;对质谱兼容的几种染色方法进行比较分析,结果表明Neuhoff胶体考染更适合于人表皮组织蛋白质的双向电泳分析,其在上样量为350μg时蛋白斑点数达394±9,匹配率为85.53%。利用基质辅助激光解析电离飞行时间质谱技术成功获得了Neuhoff胶体考染的两个蛋白斑点的肽质量指纹图谱,并鉴定为Caspase14前体和27ku热激蛋白1。【结论】建立的表皮组织双向电泳技术及其图谱可为皮肤蛋白质组学研究提供参考,为人皮肤蛋白质组学平台的构建奠定良好基础。[Objective] To establish two-dimensional electrophoresis （2-DE） technology for human epidermis, and analyze the proteome of human epidermis by mass spectrometry. [Methods] Total proteins of epidermis were extracted by homogenization and separated by 2-DE using immobilized pH gradient （IPG） strips. The 2-DE maps were matched and analyzed using image analysis software PDQuest 7.4, and some of the protein spots were identified by mass spectrometry. [Results] The 2-DE methods for proteomics analysis of human epidermis was successfully established, and four different staining 2-DE profiles were obtained and compared. Proteins spots were well distributed in pH 5 - 8 and molecular weight range of 15 ku - 100 ku. 586 ± 14 protein spots were detected in the silver staining maps. Within the three staining methods compatible with mass spectrometry, Neuhoff＇s colloidal staining was most suitable for the proteomics analysis of human epidermis. With 350μg protein loaded, 394 ± 9 protein spots were detected with a match rate of 85.53%. Finally the peptide mass fingerprinting （PMF） of two of the proteins were obtained using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, and then identified as Caspase 14 precursor and Heat shock 27ku protein 1. [Conclusion] The established 2-DE methods and maps of epidermis provide reference data for skin proteomic studies, and help to construct the platform for human skin proteomics.

关 键 词：人表皮组织 双向电泳 蛋白质组 肽质量指纹图谱 

分 类 号：Q51[生物学—生物化学]